TALEN Indel Detection Protocol

TALEN target sites were amplified from 100 ng of genomic DNA extracted using the NucleoSpin Tissue Mini Kit for DNA from cells and tissue (Macherey-Nagel) by standard PCR using Q5^® Hot Start High-Fidelity DNA Polymerase (NEB). Fragments were purified using the QIAquick PCR Purification Kit (QIAGEN) and subsequently denatured by incubation at 95°C for 5 min. Denatured DNA fragments were allowed to re-anneal through slow cooling of the samples to room temperature. Heteroduplex cleavage as surrogate readout for Indel formation/gene editing was visualized through enzymatic restriction of 100 ng of re-annealed sample with 7.5U of T7 endonuclease I (NEB) for 30 min at 37°C. T7E1 cleavage efficiency was determined by agarose gel electrophoresis and adjacent analysis of the gel images using ImageJ 1.47v.